Project Summary/Abstract: Almost all bladder cancers harbor mutations in genes involved in the regulation of chromatin state. KDM6A is among the most commonly mutated of these chromatin-modifying genes in urothelial cancers, and KDM6A alterations are by far more common in urothelial cancer than in any other human cancer type. Here, we seek to further elucidate the biologic role of KDM6A inactivation in bladder cancer pathogenesis as a prelude to the development of rational therapeutic strategies for patients with KDM6A mutant urothelial cancers. On the basis of preliminary data derived from studies of KDM6A isogenic urothelial cancer cells generated by CRISPR/Cas9-targeted inactivation, we hypothesize that KDM6A inactivation is an early event in bladder cancer pathogenesis that promotes tumor formation by deregulating genes that play key roles in modulating RAS/MAP kinase signaling. This proposal will seek to leverage recent advances in next-generation sequencing, clonality analysis, CRISPR gene editing, and patient-derived organoid and genetically-engineered mouse (GEMM) modeling to generate cellular and murine models of KDM6A-deficient bladder cancer that more accurately mirror the genomic profile, histology, and biologic properties of the human disease. These models and a unique cohort of clinically annotated human bladder cancers and the expertise of our co- investigators on this Program Project will then be used to perform in-depth analyses of the biology and functions of KDM6A in bladder cancer pathogenesis. Specifically, we will define the timing at which KDM6A alterations arise in bladder cancer (Aim 1); explore the biologic mechanisms whereby KDM6A loss promotes the transformed phenotype and their implications for drug response (Aim 2); and develop oncogene- and carcinogen-induced mouse models of bladder cancer with Kdm6a loss-of-function to study the mechanisms by which KDM6A inactivation promotes tumor initiation and/or progression in vivo (Aim 3). A long-term translational goal of this work will be to use the insights gained from studies of KDM6A mutant tumors and cell line, organoid and animal models to guide the development of novel therapeutic approaches. Integration: The proposed studies are highly complementary with Project 2, which is focused on the chromatin remodeling gene ARID1A, which is often co-altered in bladder cancers with KDM6A, and with Project 3, which is studying cancer evolution, heterogeneity, and drug response in novel patient-derived organoid models, several of which harbor KDM6A mutations. The success of this project will require close collaboration with all three Cores. Specifically, Core A will provide assistance with the processing and analysis of human tumors for Aim 1 and aid in the histologic and molecular characterization of bladder tumors that arise in GEMMs in Aim 3; Core B will provide KDM6A mutant and wildtype human organoid cell lines for Aim 2 and assist with the generation of GEMMs with targeted deletion of Kdm6a in the urothelium for Aim 3; and Core C will provide critical bioinformatic and statistical support for all Aims, and assist with program integration.